Modulated anti-VEGF therapy under the influence of lipid metabolizing proteins in Age related macular degeneration: a pilot study

Age-related macular degeneration (AMD) is a devastating retinal disease that results in irreversible vision loss in the aged population. The complex genetic nature and degree of genetic penetrance require a redefinition of the current therapeutic strategy for AMD. We aimed to investigate the role of modifiers for current anti-VEGF therapy especially for non-responder AMD patients. We recruited 78 wet AMD cases (out of 278 AMD patients) with their socio-demographic and treatment regimen. Serum protein levels were estimated by ELISA in AMD patients. Data pertaining to the number of anti-VEGF injections given (in 1 year) along with clinical images (FFA and OCT) of AMD patients were also included. Visual acuity data (logMAR) for 46 wet AMD cases out of a total of 78 patients were also retrieved to examine the response of anti-VEGF injections in wet AMD cases. Lipid metabolizing genes (LIPC and APOE) have been identified as chief biomarkers for anti-VEGF response in AMD patients. Both genotypes ‘CC’ and ‘GC’ of LIPC have found to be associated with a number of anti-VEGF injections in AMD patients which could influence the expression of B3GALTL,HTRA1, IER3, LIPC and SLC16A8 proteins in patients bearing both genotypes as compared to reference genotype. Elevated levels of APOE were also observed in group 2 wet AMD patients as compared to group 1 suggesting the significance of APOE levels in anti-VEGF response. The genotype of B3GALTL has also been shown to have a significant association with the number of anti-VEGF injections. Moreover, visual acuity of group 1 (≤ 4 anti-VEGF injections/year) AMD patients was found significantly improved after 3 doses of anti-VEGF injections and maintained longitudinally as compared to groups 2 and 3. Lipid metabolising genes may impact the outcome of anti-VEGF AMD treatment.


Methodology
Recruitments of participants. The study population comprised of 277 patients with AMD recruited from Advanced Eye Centre, PGIMER, Chandigarh, India. Analysis of Anti-VEGF response was carried out on 78 cases of active wet AMD. Although the patients were recruited prospectively, the data of 11 patients was retrieved (from same recruited patients) retrospectively to examine the number of anti-VEGF injections given in a year. Moreover, the data of visual acuity was retrieved for 46 AMD cases out of a total of 78 wet AMD patients recruited in the study. The written informed consent was obtained from all the participants after explaining the nature of study. The experimental protocols were approved by Institute Ethical Committee (IEC) (No: PGI/ IEC/2005-06; dated: 23.07.2013), PGIMER, Chandigarh, India. The study adhered to the study protocol and conducted as per the ethical guidelines laid down by Institute Ethical Committee, PGIMER, Chandigarh, India. The participants were also asked about the history of prescribed medication for any ailment along with AMD pathology. The socio-demographic (SD) details including smoking, alcohol consumption, and food habits (prior or current) etc. were also noted.
Treatment regimen of Anti-VEGF therapy. The details of a total number of anti-VEGF injections and an estimated duration of AMD pathology was obtained individually for each patient. Intravitreal Bevacizumab (1.25 mg/0.05 ml) was given to wet AMD patients. We categorised the wet AMD patients based on number of anti-VEGF injections given as described in Fig. 1. We administered three monthly doses of Bevacizumab followed by pro re nata (PRN) treatment. However, strict PRN could not be followed up in many patients owing to financial, and other logistic reasons in our part of the world.
Clinical details. Clinical severity and categorization of AMD was done by a retina specialist by recording the fluorescein fundus angiography (FFA) and optical coherence tomography (OCT) images. AREDS criteria were adopted to classify the AMD pathology in the population. Snellen's best corrected visual acuity (VA; US feet 20/20) data of 46 wet AMD patients out of the total of 78 AMD cases was collected at three time points including first (baseline), third and final visit of AMD patients along with the total visit (in months) made to the Department of Ophthalmology, PGIMER, Chandigarh. VA values were converted to logMAR scale and were considered for final data analysis. We did not take into the account the type of CNV (Classic or Occult) in our wet AMD patients. This is the limitation of our study.
Serum extraction. Blood sample of patients was collected in Sodium citrate vacutainer and kept at room temperature for 1-2 h. Samples were centrifuged at 1800 rpm for 20-30 min at room temperature. Upper layer sample (serum) were collected and stored in − 80 °C for further experimental uses. Total protein estimation. Bradford's method was adopted to estimate the total protein levels in the patient's serum. Briefly, diluted serum (600 times) was mixed with diluted Bradford's reagent (1:4 ratio). Absorbance of the reaction was taken at 595 nm using ELISA reader (BioRad, USA).

Retrospective analysis.
In order to understand the response of anti-VEGF injections in different AMD phenotypes, we retrieved the clinical data of AMD patients (n = 11) including the number of anti-VEGF shots and clinical images (both FFA and OCT) in 1 year of duration.

SNP analysis.
Single nucleotide polymorphism (SNP) analysis was carried out for lipid metabolizing genes like LIPC (rs920915) and APOE (rs769449), pro-angiogenic genes including ADAMTS9 (rs6795735) and TIMP3 (rs5749482), regulatory genes e.g. B3GALTL (rs9542236), IER3 (rs3130783), HTRA1 (rs11200638) and SLC16A8 (rs8135665, monocarboxylic transporter protein). SNP analysis was carried out on StepOne real time PCR (Applied Biosysystems Inc., Foster city, CA) by using Taq Man assay (ThermoFisher, USA) as per the manufacturer's instruction. Briefly, genomic DNA (20 ng) and 5ul of Taqman master mix was taken in the 10 μl of total volume of reaction setup. FAM and VIC tagged probes, to discriminate the allelic variation in genome at particular site, was added to the reaction. Reaction without genomic DNA was considered as negative control. Analysis of raw data to demonstrate the allelic condition (homozygous dominant/recessive and heterozygous) was performed using Genotyper and StepOne V2.0 softwares (Applied Biosysystems Inc., Foster city, CA).
ELISA. Serum levels of lipid metabolizing (APOE and LIPC), pro-angiogenic (TIMP-3 and ADAMTS9), regulatory (HTRA1, IER3 and B3GALTL) and monocarboxylic acid transporter (SLC16A8) proteins were estimated by commercially available ELISA kits (Qayee Biological Technology Co. Ltd., Shanghai, China). Serum samples were diluted before performing the experiments. The protocol was followed as per the manufacturer's instructions. Briefly, diluted serum samples were incubated with primary and secondary antibodies in dark at 37 °C for one hour. Washing was carried out 5 times, using 1X diluted washing buffer before adding the substrates to the reaction. Reaction was terminated by adding stop solution followed by estimation of absorbance at 450 nm in ELISA reader (BioRad, USA). The values were further neutralized with total protein levels for respective patients.
Statistical analysis. Comparative analysis of protein expression between various groups was estimated using One-way ANOVA, independent T-and Mann-Whitney tests. Pearson's chi square analysis was applied to reveal the association between number of anti-VEGF treatment and genotype frequency of various SNPs along with SD parameters. Logistic regression analysis was carried out to study the association of number of anti-VEGF shots and protein expression. Moreover, changes in protein expression with respect to single nucleotide polymorphism (for respective gene) were also analysed using contrast analysis with or without controlling anti-VEGF numbers. Wilcoxon sign-ranked test was employed to compare the changes in visual acuity of AMD patients throughout treatment regimen. Multivariate model analysis was performed to understand the effect of genotype interactions on anti-VEGF response (number of anti-VEGF injections per year). Survival curve was also generated for current data set in order to show direct relationship between number of anti-VEGF and progression of AMD pathology. Z-proportions test was applied to compare minor allele frequency (MAF) derived from GAW studies (INDEX-DB and IndiGenomes) conducted on Asian population with current study.

Results
Association of anti-VEGF injections with socio-demographic details. Results of chi-square suggest that alcohol addiction could be a modulator for anti-VEGF response in Indian AMD patients. Similarly, AMD patients with history of cataract surgery (single or both eyes cataract surgery) can also significantly alter the anti-VEGF response. Both results indicate the complex nature of AMD pathology where activities of daily living and associated ailment could act as a modifier for anti-VEGF response in AMD (Table 1).

Genotype influences anti-VEGF response in AMD pathology.
Chi-square analysis has revealed a significant association of B3GALTL and LIPC variants with anti-VEGF response in Indian AMD patients. Results demonstrate that the frequency of homozygous 'CC' and heterozygous 'CT' of B3GALTL are more frequent in AMD patients, being moderate and non-responsive towards anti-VEGF response with context to number of injections given to the patients. Similarly, both homozygous 'CC' and heterozygous 'GC' genotypes of LIPC are also associated with number of injections given to AMD patients (Table 2). A complex nature of AMD pathology due to its heterogeneity and genetic interaction along with equal contribution of environmental factors has been widely investigated which has also been supported by our data. However, we did not find significant association of remaining genotypes with the number of anti-VEGF injections given to the wet AMD patients (Table S1). www.nature.com/scientificreports/ study. Results of Z-test proportions did not show significant alteration of MAF between IndiGenomes and current study except HTRA1 (Table 3). Our study has indicated that response of anti-VEGF injections was found to be varied based on LIPC genotype and the level of APOE. We did not find frequencies of minor alleles of the studies genes in INDEX-DB except APOE gene which was found to be similar as frequency shown in IndiGenomes. However, references genomes from both studies haven't assessed the effect of different genotypes on anti-VEGF response or any kind of treatment strategies.

LIPC genotype influences protein expression. Associated genotypes of LIPC with anti-VEGF num-
bers have also been found to influence the majority of protein expression analysed in the study. We have demonstrated that homozygous 'CC' genotype of LIPC variant show enhanced expression of regulatory (HTRA1, B3GALTL and IER3), monocarboxylic transporter protein SLC16A8, and levels of LIPC itself. Moreover, sig-  www.nature.com/scientificreports/ nificant alteration of protein expression, including HTRA1, IER-3 and LIPC, has also been examined in heterozygous 'GC' genotype of LIPC variants (Fig. 2). However, we did not find significant alteration of proteins among B3GALTL genotypes which has also showed the association with number of anti-VEGF injection in AMD patients (Table 2). Similarly, the expression of studied proteins were not found to be significantly altered with reference to other genotypes except the SLC16A8 expression between ' AA' and 'GA' genotypes of HTRA1 (Table S2). Additionally, contrast estimate indicated significant changes in LIPC levels by 17.578 pg/ug with alteration of genotype i.e. from 'GG (reference genotype)' to 'CC' genotype (p = < 0.0001) which is consistent with our previous results 15 (Table 4). Interestingly, we did not find any significant alteration for any other protein levels against the changes in genotypes (of studied variants) while considering anti-VEGF number as covariate. Results   www.nature.com/scientificreports/ show an indirect role of lipid metabolism by regulating the action of associated proteins (LIPC) in controlling the anti-VEG response. Results also signify the biological significance of particular genotype (of variants), genetic and allelic interactions under the influence of confounders which may influence the various protein expressions thereby modulating the AMD treatment outcome after anti-VEGF.

APOE mediated anti-VEGF response in AMD. Enhanced APOE levels with successive anti-VEGF
injections (≥ 5 of per year) in AMD patients have suggested the APOE dependent anti-VEGF response in Indian AMD (Fig. 3). Significantly elevated expression of APOE has been observed in moderate group (group 2; ≥ 5 anti-VEGF injections/year and continuing for < 36 months) as compared to mild group (group 1; ≤ 4 anti-VEGF injections/year). Similarly, APOE levels were also found to be higher in severe group (group 1; ≥ 5 anti-VEGF/ year and continuing for > 36 months) as compared to mild group of AMD, though it was not statistically significant. Results suggested that lipid metabolizing genes (especially APOE and LIPC) may modulate the action of anti-VEGF in AMD pathology.
To further validate the results suggesting the role of lipid metabolizing genes in anti-VEGF response, we assessed the scale of anti-VEGF injections given to AMD patients (for 11 AMD patients, Fig. 4). Pearson's correlation analysis has revealed the positive correlation between anti-VEGF treatment and expression of ADAMTS9 (PCC = 0.629; P = 0.020), APOE (PCC = 0.872; P = < 0.0001) and SLC16A8 (PCC = 0.656; P = 0.014). Response   Table 5). Results suggest that APOE and LIPC may act as chief modulator for anti-VEGF treatment in AMD patients. When we compared the visual acuity data among studied groups, significant improvement of visual acuity from baseline was observed in group 1 AMD cases after three doses of anti-VEGF treatment as compared to group 2 and group 3. However, visual acuity was also improved in case of group 2 and 3 AMD cases after 3 doses of anti-VEGF treatment but it was non-significant. Number of anti-VEGF injections were further correlated with visual acuity (VA) of group-wise AMD patients along with their total follow up. Results have also shown that while comparing final visual acuity of group 2 and 3, AMD cases within group 1 worsened. Longitudinal follow-up of patients revealed more consistent results of visual acuity examined in group 1 AMD patients as compared to group 2 and group 3 ( Table 6). This may require more anti-VEGF injections to stabilize the visual acuity as in case of group 2 and 3 in our results.

Influence of genetic interaction on anti-VEGF response.
Our results have shown the role of lipid metabolizing genes in modulating anti-VEGF response in AMD pathology. Hence, we further attempted to assess the impact of genetic interaction on anti-VEGF response in AMD. The analysis of data revealed a significant genotype interaction among ADAMTS9-TIMP3 genes in AMD pathology. However, we did not find direct influence of genotype interaction on response of anti-VEGF treatment (in terms of number of injections given) and association with disease progression (Table 7). Results also suggest that studied SNP variants and their genetic interactions, especially among pro-angiogenic genotypes (ADAMTS9-TIPM3), may exacerbate the AMD pathology suggesting an indirect implication of the same on anti-VEGF response.
We wanted to examine the progress of disease in patients as with the duration of disease (in months), such as the effect of anti-VEGF treatment, until the occurrence of the AMD pathology. For this purpose, survival analysis was performed and Kaplan-Meier survival curve revealed that at 12 months anti-VEGF treatment can provide 64% symptomatic recovery from AMD, while at 36 months, it was only 25% (Fig. 5). Subsequently, symptomatic relief from AMD by anti-VEGF treatment waned in patients receiving the successive anti-VEGF treatment with gradual increase in number of injections (anti-VEGF). This may be due to uncontrolled activity of lipid metabolizing proteins under the influence of confounders along with the genetic complexity of an individual 15 . Moreover, we have also determined the median survival time by locating the (time 'in months'), at which the cumulative survival proportion is 0.5. In our study, median survival rate due to the effect of anti-VEGF treatment is 18 months with standard error of 1.849 and confidence intervals (14. 38-21.63) (Fig. 5). www.nature.com/scientificreports/

Discussion
The need for personalized medicine cannot be emphasised unless the genetics and nature of interactions with genetic variants and environmental factors well understood which acts as a roadblock towards translational approach in AMD genetics 18 . This study has attempted to understand the unique outcome of anti-VEGF treatment under the influence of confounders and genetic variants. We have shown the outcome of anti-VEGF (in context to number of injections given during the disease course) associated with both environmental (alcohol consumption and cataract history) and genetic factors (genetic variants of B3GALTL and LIPC). Poor response of Aflibercept has also been observed with higher BMI and geographic atrophy AMD patients 19 . Aqueous humor levels of angiogenic and pro-angiogenic proteins including VEGF-A, VEGF-C, interleukin 8, endothelin 1, HGF (Hepatocyte growth factor), HB-EGF (Heparin-binding epidermal growth factor-like growth factor), follistatin, and angiopoietin 2 were also found to be elevated after intravitreal injection of bevacizumab 20 . ATG haplotype of rs699947 (− 2578 C/A), rs2010963 (+ 405 C/G) and rs3025039 (+ 936 C/T) SNPs has been earlier shown to be associated with 'poor' responder of intravitreal bevacizumab in Tunisian AMD Patients 21 . Our results suggest that VEGF could be a potential identifier for anti-VEGF response by considering the lipid metabolizing genes as a modifier (especially APOE and LIPC) which is consistent with our previous report in the field 12 .
Recently, TT genotype of CFH genetic variant (Y402H) was shown to increase the function and response of intravitreal ranibizumab in AMD patients 22 . Interestingly, a significant alteration in LIPC (lipid metabolizing), TIMP-3 (angiogenic) and SLC16A8 (monocarboxylic transporter) was observed in CFH negative AMD cases 23 .
Our results have also revealed the association of genetic variants of B3GALTL and LIPC with the number of anti-VEGF injections in Indian AMD patients. Moreover, we also found a significant differential expression of B3GALTL, HTRA1, IER3 and LIPC proteins among subgroups of LIPC genotype. Genetic interaction of various genotypes can also influence the outcome of anti-VEGF treatment in AMD pathology. We have demonstrated Table 7. Multivariate analysis to demonstrate genotype interaction of studied SNPs (based on their cellular functions) and influence of anti-VEGF treatment on AMD pathology. Results showed significant genotype interaction of pro-angiogenic genes including ADAMTS9 (rs6795735) and TIMP3 (rs5749482), but didn't show direct influence of genotype interactions on number of anti-VEGF injections in Indian AMD patients. www.nature.com/scientificreports/ a significant interaction between pro-angiogenic ADAMTS9-TIMP3 genotypes. However, we did not find significant association between number of anti-VEGF injections and such genetic interaction studied in our population. This indicate a complex nature of AMD pathology and associated response of anti-VEGF treatment which can be dependent on the nature of genetic interaction along with contribution of confounders 24 . Moreover, our results have also showed that both APOE and LIPC may act as biomarkers to differentiate degree of anti-VEGF response in wet AMD cases with respect to number of anti-VEGF injection given to the patients. The treatment strategy for lipid metabolism (by targeting APOE and/or LIPC) along with anti-VEGF may be a crucial step for effective management of AMD. Results of visual acuity and changes VA after anti-VEGF treatment have suggested the group 1 as a responder in comparison to group 2 and 3 where anti-VEGF treatment did not lead to significant changes in VA (especially after 3 doses of anti-VEGF injections). Out results of visual acuity and number of anti-VEGF injections have further supported the hypothesis of current study which indicates subsequent changes in number of anti-VEGF injections (or response) and visual acuity outcome based on genetic susceptibility of AMD patient. Conclusively, results indicate the prominent biological significance of lipid metabolizing molecules (including APOE and LIPC) which may influence the anti-VEGF outcome in AMD patients. Impact of genetic variants and their interaction cannot be ignored in modulating the anti-VEGF response which must be considered for redefining the management of AMD pathology. However, conclusion of this study was drawn on limited number of samples along with number of anti-VEGF injections. Visual acuity of anti-VEGF treated groups has also suggested that group 1 AMD patients (≤ 4 anti-VEGF injections/year) respond to anti-VEGF treatment and showed more persistent visual acuity as compared to group 2 (≥ 5 anti-VEGF injections/year till < 36 months) and 3 (≥ 5 anti-VEGF injections/year for > 36 months). Final visual acuity of group 2 and 3 have further deteriorated than group 1 AMD cases indicating the longitudinal implication of genetic susceptibility (especially through LIPC and APOE) and response towards anti-VGEF treatment (also the number of anti-VEGF injections). This study could serve as substrate to design larger study on geographically diverse range of population based on their genetic susceptibility, genetic interactions, penetrance and influence of environmental factors.

Data availability
Whole data can be provided by first and corresponding authors of the manuscript without any restriction whenever required.